The long-term aim of his research is to identify genetic predispositions to complications of alcoholism. The immediate goal for this grant period is to elucidate the basis of an abnormality in the thiamin pyrophosphate (TPP)-dependent enzyme transketolase (EC 2.2.1.1). An impaired affinity to TPP (high Km) has been postulated to predispose a subpopulation of alcoholics to the development of a form of thiamin deficiency, the Wernicke-Korsakoff's syndrome. Structural and molecular genetic studies is proposed to characterize the aberrant transketolase in cultured skin fibroblasts from patients with Wernicke-Korsakoff's syndrome or from patients with documented transketolase abnormality. Structural comparisons will be performed by immunoblots of electrophoretic gels (SDS-gels, isoelectric focusing gels and native gels), by activity staining of electrophoretic gels, by peptide mapping and other immunochemical techniques. This proposition will also examine the possibility of multiple forms of human transketolase. Preliminary work has already purified human liver transketolase, raised antibodies and standardized immunochemical procedures. For molecular genetic studies, cDNA for normal human transketolase will be isolated (with the antibody probe) sequenced, and used to isolated genomic DNAs to construct a structural map for the transketolase gene. The cDNA for aberrant transketolase will also be isolated and sequenced to identify the exact site of genetic mutation. The mutation will be further tested at the genomic level using the abnormal genomic fragment amplified with the polymerase chain reaction (PCR) method. These studies will answer the following 2 questions. Is the aberration of Wernicke-Korsakoff transketolase associated with a structural abnormality? If so, is this structural abnormality determined at the genetic level?